PROJECT SUMMARY Project 3 will test the hypothesis that identification of the transmitted founder and mutated, sequential SHIV Env glycoproteins that bind germline or evolved V1V2 or V3-glycan broadly-reactive neutralizing antibody (bNAb)-specific B cell receptors (BCRs) will provide immunogens that will initiate similar bNAb lineages in rhesus macaques (RM) in the setting of vaccination. Soluble native-like Env trimers, glycopeptides, and/or DNA vectored env gp160 construct immunogens will be produced to determine the optimal immunogen forms required for initiation and elicitation of V1V2- and V3-glycan targeted Nabs. In this project, sequential Env immunization regimens will be designed in collaboration with Projects 1 and 2 and Cores B (Sequencing) and C (Bioinformatics and Statistics). Protection following vaccination will be assessed using autologous and heterologous SHIVs from Project 1. Specific aims of Project 3 are: 1. To express autologous TF and evolved Envs from SHIV-infected RM as recombinant gp120s and as stabilized trimers; 2. To perform binding assays of bNAb lineage BCRs with evolved autologous and heterologous Envs and, in conjunction with the Bioinformatics and Statistics Core, to design sequential Env immunogens based on binding affinity and/or on mutations inferred bioinformatically to be associated with bnAbs recognition; 3. To perform immunization studies of Env constructs in bNAb germline humanized mice and RM, including: (a) B cell repertoire analysis and plasma neutralization assays on vaccinated mice and RMs in collaboration with Project 2, (b) sequential immunizations with adjuvants and checkpoint inhibitor administration to promote bNAb lineage survival and maturation, and (c) negative-stained EMs and vaccine-induced antibody-Env co-crystallization studies of vaccine-induced antibodies. Project 3 will synergize with Projects 1 and 2 and Cores B and C to complete the pre-clinical translational pipeline for developing HIV vaccine strategies based on recapitulating the virologic and immunologic events that occur in bNAb development during transmitted/founder Env SHIV infection in RMs.